Methods and devices for hybridization and binding assays using thermophoresis

ABSTRACT

An apparatus and method for performing hybridization or binding assays under thermophoretic conditions is provided.

BACKGROUND OF THE INVENTION

[0001] This invention relates to methods for improving thediscrimination of hybridization of target molecules to probes onsubstrate-bound oligonucleotide, peptide, or protein arrays. Therefore,it relates to the fields of molecular biology and biophysics.

[0002] An efficient method of sequencing DNA is by means ofhybridization to known nucleotide sequences arranged in microarrays. Seee.g., PCT WO 89/10977. In this method a solution of single strands ofunknown DNA is mixed with an array of oligonucleotides which are fixedto a substrate. The oligonucleotides vary in sequence, and each uniquesequence occupies a small region on the substrate whose position isknown. If the sequence of a given oligonucleotide region iscomplementary to the unknown DNA sample, then the DNA strands willhydrogen bond or hybridize to the oligonucleotides at that site. Sincethe oligonucleotide sequence is known, that part of the sample whichbound to the oligonucleotide is then determined as well. If the DNAsample is fragmented into lengths comparable to the lengths of theoligonucleotides, then the entire DNA sample can, in principle, besequenced.

[0003] The sites in the microarray at which the DNA binds can beidentified by attaching tags to the sample fragments beforehybridization. These tags can be radioactive, fluorescent, orluminescent, for example. By scanning the hybridized microarray forradioactivity or fluorescence, the hybridized sites can be identified.

[0004] The power of this technology lies in the discriminatory abilityof the hybridization process. For DNA fragments on the order of 20nucleotides in length or less, a single mismatch in nucleotide basepairs can significantly affect the hybridization process, and more thanone adjacent base pair mismatch can effectively prevent hybridization.The degree of discrimination is controlled by the conditions in thesolution: the types and concentrations of buffers and the temperature.The degree to which nucleic acids hybridize is referred to as“stringency”. In a state of high stringency conditions, hybridizationrate is reduced and the probability of base pair mismatches is reducedeven more. In a condition of low stringency, hybridization becomes morelikely and the probability of base pair mismatches increases. Ingeneral, high stringency conditions and high discrimination against basepair mismatches are characterized by higher temperature, lower ionicstrength, low reactant concentration, and short reaction times. Inaddition, many washings of the microarray with hybridization buffers aredone to remove sample DNA strands which have not hybridized to probeoligonucleotides.

[0005] The initial DNA sample is often very limited in size, and toincrease the probability of detecting a successful hybridization in themicroarray, the DNA is amplified using polymerase chain reaction (PCR)or other means. Despite the amplification process, the DNA concentrationis often still very limited, so hybridization of a substantial fractionof the sample may be needed for reliable detection. Thus conditions ofhigh stringency may also limit the detectability of hybridized samples.

[0006] Hybridization rates in the microarray are ultimately limited bydiffusion of the DNA samples in their buffer to the substrate. Morespecifically, the microarray is mounted within a structure (i.e., acell) which serves as a reservoir for the DNA sample. Various techniquesare used to circulate the samples within the cell to expeditehybridization, such as circulation of the sample from the cell to anexternal reservoir and back, or by agitation of the cell, buthybridization times can still be many hours. Furthermore, washing themicroarray in buffer to remove DNA samples which did not hybridize tooligonucleotide sites, thereby increasing the stringency conditions, cantake a comparable amount of time. See, for example, U.S. Pat. No.6,114,122.

[0007] One technique to speed things up is to use eletrophoresis toattract the negatively charged DNA samples to the oligonucleotides. Thisrequires adding electrodes and an electrical grid to the microarray, sothat an electric field with the right polarity can be established toattract the DNA to the oligonucleotides. The electrical mobility of theDNA can be much greater than the intrinsic diffusion rate in solution.After hybridization has taken place, the polarity of the field can bereversed, thereby driving the non-hybridized DNA samples away from themicroarray, and making the washing steps more effective. This cangreatly increase the stringency of the process while reducing theoverall hybridization time. See, e.g., U.S. Pat. No. 5,849,486.

[0008] However, these improvements are purchased at the expense of addedcomplexity. The microarray must be provided with an electrical grid.Moreover, the grid must be covered by a permeation layer which isolatesand protects the DNA from the metallic grid, excludes electrolysisproducts from the DNA buffer, and provides support for theoligonucleotide probes. High density microarrays are typically scannedfor fluorescence through their transparent substrates. This is notpossible if an electrical grid is present. The buffer properties must beadjusted to accommodate the electrophoresis. In particular, the bufferelectrical conductivity must not be low. These constraints may notpermit using buffers which are optimal for hybridization.

[0009] The concept of a microarray to identify unknown samples can beextended to other molecules such as proteins. In the technique known asELISA (enzyme linked immunosorbent assay), an array of known antibodiesis created on a substrate. The array is then exposed to a solution ofunknown proteins (i.e., antigens). After washing, proteins which remainbound to their corresponding antibodies can be fluorescently tagged, andidentified from their locations in the array. While electrophoresis canbe used here, the variety of charge states of different proteinscomplicates the experiments.

[0010] Temperature gradient gel electrophoresis, as described in GermanApplication DE-OS 36 22 591, is a method for detecting slight structuraldifferences or peculiarities of biological macro-molecules such asnucleic acids or proteins. This technique relies upon the use oftemperature gradients in combination with electrophoresis for theseparation of biological macro-molecules. This technique, however, isrestricted to the operation of flat-bed gel electrophoresis.

[0011] Thermophoresis refers to a process in which particles, residingin a gas supporting a temperature gradient, are driven away from warmsurfaces toward cooler surfaces. The thermophoretic drift velocity isfound to be directly proportional to the temperature gradient in thegas. Although the phenomenon has been well studied with aerosols, therehas been little reported on the use of thermophoresis with particles inliquid. McNab et al. (1973) J. Colloid and Interface Science 44:339 havepresented an equation describing thermophoretic drift velocity based ona study of small latex spheres in water and hexane. No dependence onparticle size was detected within the limited particle size rangestudied. The thermophoretic drift velocity was found to be directlyproportional to the temperature gradient in the fluid.

[0012] Thus, there is still a need for methods for improving thestringency of and/or decreasing the time required for hybridizationexperiments. The present invention addresses this and other needs.

SUMMARY OF THE INVENTION

[0013] The present invention provides a method for performing ahybridization assay between a target nucleic acid molecule and anoligonucleotide array. The array comprises surface to which arecovalently attached oligonucleotide probes with different, knownsequences, at discrete, known locations. The method comprises the stepof contacting or incubating the array with a hybridization mixturecomprising the target, and optionally an isostabilizing agent, underthermophoretic conditions and determining the identity of probes towhich the target has hybridized. Preferably, the thermophoreticconditions comprise the application of a temperature gradientperpendicular to the array surface whereby the target is driven to thearray surface. The method also may further comprise the step ofreversing the temperature gradient, whereby any unhybridized target isdriven away from the array surface.

[0014] Preferably, the target further comprises a detectable label andthe array has a density of at least ten thousand features per square cm.In one embodiment, the array surface is vertical and the temperaturegradient is horizontal. In another embodiment, the array surface ishorizontal and the temperature gradient is vertical. Preferably, atemperature gradient of about 10° C./mm is used.

[0015] The invention also provides a method for performing ahybridization assay between a target nucleic acid molecule and anoligonucleotide array, the array comprising a surface to which arecovalently attached oligonucleotide probes with different, knownsequences, at discrete, known locations, wherein such probes have beencontacted with a hybridization mixture comprising the target nucleicacid molecule. The method comprises the steps of: applying a temperaturegradient to the array surface whereby any unhybridized target is drivenaway from the array surface; and determining the identity of probes towhich the target has hybridized.

[0016] A method for performing a binding assay between a target moleculeand an array is also provided. According to this embodiment, the arraycomprises a surface to which are covalently attached a plurality ofbinding partners with different, known sequences, at discrete, knownlocations. The method comprises the steps of: incubating the array witha mixture comprising the target under thermophoretic conditions; anddetermining the identity of binding partners to which the target hasbound.

[0017] Apparatus for performing the various methods described above arealso provided. The apparatus preferably will comprise a containerconnected to at least one temperature control blocks in aheat-conducting fashion, such that a temperature gradient is produced.The container may be connected to two temperature control blocks in aheat-conducting fashion and may further comprise an inlet port and anoutlet port.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 shows an embodiment of the invention wherein ahybridization cell oriented horizontally is installed between twotemperatures reservoirs.

[0019]FIG. 2 shows an embodiment of the invention wherein ahybridization cell oriented vertically is installed between twotemperature reservoirs.

[0020]FIG. 3 illustrates a free convection circulation flow that mayoccur when the hybridization cell is oriented as shown in FIG. 2.

DETAILED DESCRIPTION OF THE INVENTION

[0021] General

[0022] It is to be understood that the terminology used herein is forthe purpose of describing particular embodiments only and is notintended to limit the scope of the present invention which will belimited only by the appended claims. It must be noted that as usedherein and in the appended claims, the singular forms “a”, “an”, and“the” include plural reference unless the context clearly dictatesotherwise. Unless defined otherwise, all technical and scientific termsused herein have the same meanings as commonly understood by one ofordinary skill in the art to which this invention belongs. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention, thepreferred methods, devices, and materials are now described.

[0023] Definitions

[0024] “Complementary” refers to the topological compatibility ormatching together of interacting surfaces of a probe molecule and itstarget. Thus, the target and its probe can be described ascomplementary, and furthermore, the contact surface characteristics arecomplementary to each other.

[0025] “Denaturing agent” refers to compositions that lower the meltingtemperature of double stranded nucleic acid molecules by interferingwith hydrogen bonding between bases in a double-stranded nucleic acid orthe hydration of nucleic acid molecules. Denaturing agents can beincluded in hybridization buffers at concentrations of about 1 M toabout 6 M and, preferably, about 3 M to about 5.5 M.

[0026] “Denatured nucleic acid” refers to a nucleic acid which has beentreated to remove folded, coiled, or twisted structure. Denaturation ofa triple-stranded nucleic acid complex is complete when the third strandhas been removed from the two complementary strands. Denaturation of adouble-stranded DNA is complete when the base pairing between the twocomplementary strands has been interrupted and has resulted insingle-stranded DNA molecules that have assumed a random form.Denaturation of single-stranded RNA is complete when intramolecularhydrogen bonds have been interrupted and the RNA has assumed a random,non-hydrogen bonded form.

[0027] “Feature” refers to an area of a substrate having a collection ofsame-sequence, surface-immobilized molecules. One feature is differentthan another feature if the probes of the different features havedifferent sequences of component molecules.

[0028] “Fluorescence probe molecule” refers to a fluorophore, which is afluorescent molecule or a compound which is capable of binding to anunfolded or denatured receptor and, after excitement by light of adefined wavelength, emits fluorescent energy. The term fluorescenceprobe molecule encompasses all fluorophores. More specifically, forproteins, the term encompasses fluorophores such as thioinosine, andN-ethenoadenosine, formycin, dansyl derivatives, fluoresceinderivatives, 6-propionyl-2-(dimethylamino)-napthalene (PRODAN),2-anilinonapthalene, and N-arylamino-naphthalene sulfonate derivativessuch as 1-anilinonaphthalene-8-sulfonate (1,8-ANS),2-annilinonaphthalene-6-sulfonate(2,6-ANS),2-aminonaphthalene-6-sulfonate,N,N-imethyl-2-aminonaphthalene-6-sulfonate, N-phenyl-2-aminonaphthalene,N-cyclohexyl-2-aminonaphthalene-6-sulfonate,N-phenyl-2-aminonaphthalene-6-sulfonate,N-phenyl-N-methyl-2-aminonaphthalene-6-sulfonate,N-(o-toluyl)-2-aminonaphthalene-6-sulfonate,N-(m-toluyl)-2-aminonaphthalene-6-sulfonate,N-(p-toluyl)-2-aminonaphthalene-6-sulfonate,2-(p-toluidinyl)-naphthalene-6-sulfonic acid (2,6-TNS), 4-(dicyanovinyl)julolidine (DCVJ), 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN),6-hexadecanoyl-2-(((2-(trimethylammonium)ethyl)methyl)amino)naphthalenechloride(PATMAN), nile red, N-phenyl-1-naphthylamine,1,1-dicyano-2-[6-dimethylamino) naphthalen-2-yl]propene (DDNP),4,4′-dianilino-1,1-binaphthyl-5,5-disulfonic acid (bis-ANS), andDAPOXYL.TM. derivatives (Molecular Probes, Eugene, Oreg.). Preferablyfor proteins, the term refers to 1,8-ANS or 2,6-TNS. Another type offluorophore is a semiconductor nanocrystal, several nanometers indiameter, whose fluorescent properties are affected by quantumconfinement effects. Such crystals are described in e.g. U.S. Pat. No.5,990,479.

[0029] “Hybridization optimizing agent” refers to a composition thatdecreases hybridization between mismatched nucleic acid molecules, i.e.,nucleic acid molecules whose sequences are not exactly complementary.

[0030] “Incubating ” refers broadly to placing the target moleculeand/or hybridization mixture in contact with the array. Preferably,incubating refers to the equilibration of binding between the targetmolecule and the substrate-bound molecule to be tested for binding.

[0031] “Isostabilizing agent” refers to a composition that reduces thebase-pair composition dependence of DNA thermal melting transitions.More particularly, the term refers to compounds that, in properconcentration, result in a differential melting temperature of no morethan about 1 C for double stranded DNA oligonucleotides composed of ATor GC, respectively. Isostabilizing agents preferably are used at aconcentration between 1 M and 10 M, between 2 M and 6 M, between 4 M and6 M, between 4 M and 10 M and, optimally, at about 5 M. Betaines andlower tetraalkyl ammonium salts are examples of isostabilizing agents.See, U.S. Pat. No. 6,045,996, which is incorporated herein by reference.

[0032] “Oligonucleotide array” refers to a substrate having a surfacehaving at least two different features. Oligonucleotide arrayspreferably have a density of at least five hundred, at least onethousand, at least 10 thousand, at least 100 thousand, at least onemillion or at least 10 million features per square cm. In oneembodiment, the arrays have a density of about 625 features per squarecm. The substrate can be, merely by way of example, silicon or glass andcan have the thickness of a glass microscope slide or a glass coverslip. Substrates that are transparent to light are useful when themethod of performing an assay on the chip involves optical detection. Asused herein, the term also refers to a probe array and the substrate towhich it is attached that form part of a wafer.

[0033] “Probe” refers to a surface-immobilized molecule, e.g., anoligonucleotide, peptide, or protein, that can be recognized by aparticular target. Depending on context, the term “probe” refers both toindividual molecules and to the collection of same-sequence moleculessurface-immobilized at a discrete location.

[0034] “Renaturation accelerant” refers to compounds that increase thespeed of renaturation of nucleic acids by at least 100-fold. Theygenerally have relatively unstructured polymeric domains that weaklyassociate with nucleic acid molecules. Accelerants include heterogenousnuclear ribonucleoprotein (“hnRP”) Al and cationic detergents such as,preferably, CTAB (“cetyltrimethylammonium bromide”) and DTAB (“dodecyltrimethylammonium bromide”), and, also, polylysine, spermine,spermidine, single stranded binding protein (“SSB”), phage T4 gene 32protein and a mixture of ammonium acetate and ethanol. Renaturationaccelerants can be included in hybridization mixtures at concentrationsof about 1 μ.M to about 10 mM and, preferably, 1 μ.M to about 1 mM. TheCTAB buffers work well at concentrations as low as 0.1 mM.

[0035] “Screening” refers to the testing of a multiplicity of moleculesor compounds for their ability to bind to a target molecule which iscapable of denaturing.

[0036] “Target” refers to a nucleic acid molecule or protein that has anaffinity for a given probe. Targets may be naturally-occurring orman-made nucleic acid molecules or proteins. Also, they can be employedin their unaltered state or as aggregates with other species. Targetsmay be attached, covalently or noncovalently, to a binding member,either directly or via a specific binding substance. Targets aresometimes referred to in the art as anti-probes. A “Probe-Target Pair”is formed when two macromolecules have combined through molecularrecognition to form a complex.

[0037] Hybridization Assays

[0038] Hybridization assays on substrate-bound oligonucleotide arraystypically involve a hybridization step and a detection step. In thehybridization step, a hybridization mixture containing the target andoptionally, an isostabilizing agent, denaturing agent or renaturationaccelerant is brought into contact with the probes of the array andincubated for a time appropriate to allow hybridization between thetarget and any complementary probes. Usually, unbound target moleculesare then removed from the array by washing with a wash mixture that doesnot contain the target, such as hybridization buffer. This leaves onlybound target molecules. In the detection step, the probes to which thetarget has hybridized are identified. Since the nucleotide sequence ofthe probes at each feature is known, identifying the locations at whichtarget has bound provides information about the particular sequences ofthese probes.

[0039] The hybridization mixture includes the target nucleic acidmolecule and, optionally, a hybridization optimizing agent in anappropriate solution, i.e., a hybridization buffer. The target nucleicacid molecule is present in the mixture at a concentration between about0.005 nM target per ml hybridization mixture and about 50 nM target perml hybridization mixture, preferably between about 0.5 nM/ml and 5 nM/mlor, more preferably, about 1 nM/ml and 2 nM/ml. The target nucleic acidmolecule preferably includes a detectable label, such as a fluorescentprobe molecule. Additional examples of hybridization conditions areprovided in several sources, including: Sambrook et al., MolecularCloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N.Y.;and Berger and Kimmel, “Guide to Molecular Cloning Techniques,” Methodsin Enzymology, (1987), Volume 152, Academic Press, Inc., San Diego,Calif.; Young and Davis (1983) Proc. Natl. Acad. Sci. (U.S.A.) 80: 1194.

[0040] The hybridization mixture is placed in contact with the array andincubated. Contact can take place in any suitable container, forexample, a dish or a flow cell specially designed to hold the array andto allow introduction of the fluid into and removal of it from the cellso as to contact the array. In a preferred embodiment, the container hasa volume from approximately 50 to approximately 500 microliters. Inorder to achieve large temperature gradients, to increase thethermophoretic drift velocity, without recourse to excessive temperaturedifferences, the gap between the probe surface and the opposed surfaceis preferably kept to a minimum. In a preferred embodiment, this gap iskept to 1 mm or less. The small gap also reduces the time needed for theanalyte to drift to the reactive substrate.

[0041] The present invention generally incorporates temperaturemonitoring and control systems for optimization of hybridizationconditions. Temperature control may be carried out by a variety ofmeans. For example, a temperature control block may be placed adjacentto at least one of the external surfaces of the container. Preferably,the temperature gradient may be built up in such fashion that, using acontrollable temperature control block, a specified temperature level isadjusted at one side of the container, while a second, spatiallyseparated temperature level is defined by the temperature level at theopposite side. Thus, the container is connected to at least one, andpreferably to two, temperature control blocks in a heat-conductingfashion.

[0042] According to one embodiment the substrate is horizontal (FIG. 1)and a vertical temperature gradient is created with the appropriatedirection whereby the thermophoretic force will thus drive the DNAsamples toward the lower probe surface. Circulation or agitation of thefluid may be needed for lateral redistribution.

[0043] When the lower surface is warmer than the upper surface, onemight expect formation of Rayleigh-Benard convection cells, whereinbuoyancy forces drive warm liquid from the bottom of the cell up to thecooler surface, where it cools, becomes more dense, and sinks back Thismay affect either the hybridization rate or the achievable stringency.However the onset of the Rayleigh-Benard instability is retarded whenthe separation between the upper and lower surfaces is small, and whenthe temperature difference is small. Therefore Rayleigh-Benardconvection may be absent in some embodiments of this invention.

[0044] In another embodiment, the cell is oriented vertically (FIG. 2)and some free convection will take place. The circulation will probablybe similar to that shown in FIG. 3. This flow, which includes the DNAmolecules, is superimposed on the horizontal thermophoretic drift of theDNA toward or away from the oligonucleotide substrate. The freeconvection flow may assist lateral redistribution of the DNA molecules.

[0045] The desired temperature gradient is maintained within thecontainer by thermal exchange across the relatively thin walls of thecontainer against which one or more temperature controller blocks areplaced. The thickness of the wall is typically dependent upon a numberof factors including, e.g., the composition of the material, the desiredtemperature range, the thermal conductivity of the wall, manufacturingtolerances, and the like.

[0046] Generally, incubation will be at temperatures normally used forhybridization of nucleic acids, for example, between about 20° C. andabout 75° C, e.g., about 25° C., about 30° C., about 35° C., about 40°C., about 45° C., about 50° C., about 55° C., about 60° C. or about 65°C. For probes longer than about 14 nucleotides, 37° C.-45° C. ispreferred. For shorter probes, 55° C. -65° C. is preferred.

[0047] Preferably, a temperature gradient of between about 5 and 25°C./mm, more preferably, between about 5 and 15° C./mm, and mostpreferably, a temperature gradient of about 10° C./mm will be used.

[0048] The target is incubated with the probe array for a timesufficient to allow the desired level of hybridization between thetarget and any complementary probes in the array. Using a hybridizationtemperature of 25° C. and with a 10° C./mm temperature gradient yields avery clear signal, usually in at least 30 minutes to two hours, but itmay be desirable to hybridize longer, i.e., about 15 hours.

[0049] In preferred aspects, the temperature control block may be athermoelectric temperature controller, e.g., a Peltier heater/cooler.Alternatively, a temperature control block may incorporate a series ofchannels through which is flowed a recirculating temperature controlledfluid, e.g., water, ethylene glycol or oil, which is heated or cooled toa desired temperature, e.g., in an attached water bath. The temperaturecontrol blocks may be time-controllable.

[0050] Exemplary heating elements include resistive heaters, such as an80 W HD04-0100N heater available from Heater Cartridge, Wooddale, Ill.Exemplary cooling elements includes Peltier crystals (which can cool theplate to about −20 degrees C.), frozen substances, and the like. Oneexemplary Peltier device is model TE 9501/127/030B, available fromMelcor Thermoelectrics. One or more water baths capable of beingthermostatted can also be used as a heating or cooling element. Suchelements may be placed directly against the container or within athermally conductive material which is in direct contact with thecontainer.

[0051] The system may also include a temperature control element topreheat or precool fluids prior to injection into the container. Forexample, the reagent/sample vessels may be placed in a temperaturecontrolled environment, e.g., a water bath, to achieve optimalpre-injection temperatures. Alternatively, an in-line temperaturecontroller may be employed to adjust the temperature of the fluid as itis being delivered to the container. Typically, this involves the use ofa coiled heat-exchange tube as part of the fluid passage. Theheat-exchange coil is generally disposed around a temperature controlledelement and is fabricated from a material having a relatively highthermal transfer coefficient, e.g., stainless steel, copper, aluminum,etc.

[0052] The container preferably includes an aperture which permitsoptical access to the array. The aperture or window may be a quartz, orother suitable material chose in part for its transmission andnon-fluorescence properties. Advantageously, the window is chosen tohave an index of refraction which substantially matches the index ofrefraction of the sample solution.

[0053] An inlet port and an outlet port may be provided through the flowcell. An input tube is preferably connected to the inlet port.Optionally, the input tube connects to a fluidic interface port, such asformed by a female Luer taper system. An output tube is preferablyconnected to the outlet port. The components of the fluidic system arepreferably formed from inert materials, e.g., tetrafluoroethylene, orother medical grade plastics. The flow cell and associated componentsmay be formed through any known technique, such as molding or machining.The output tube preferably provides a communication path from the flowcell to a reservoir.

[0054] After the desired reaction is complete, the array usually iswashed with the hybridization buffer, which also can include thehybridization optimizing agent. These agents can be included in the samerange of amounts as for the hybridization step, or they can beeliminated altogether. Preferably, the temperature gradient is alsoreversed thereby creating a thermophoretic force in the directionopposite to the prior attractive force. In this way, nonspecificanalytes or unreacted molecules may be removed from the array. Specificanalytes or reaction products may be released from any array or featurethereof and transported to other locations for further analysis; orstored at other addressable locations; or removed completely from thesystem. This removal or deconcentration of materials by reversal of theforce enhances the discrimination ability of the system by resulting inremoval of nonspecifically bound materials. By controlling the amount ofnow repulsive thermophoretic force to nonspecifically bound materials onthe array, stringency control may be achieved. By increasing thetemperature gradient so as to remove partially hybridized DNA sequences,thereby permitting identification of single mismatched hybridizations,point mutations may be identified.

[0055] Preparation of Target Samples

[0056] The target polynucleotide whose sequence is to be determined isusually isolated from a tissue sample. If the target is genomic, thesample may be from any tissue (except exclusively red blood cells). Forexample, whole blood, peripheral blood lymphocytes or PBMC, skin, hairor semen are convenient sources of clinical samples. These sources arealso suitable if the target is RNA. Blood and other body fluids are alsoa convenient source for isolating viral nucleic acids. If the target ismRNA, the sample is obtained from a tissue in which the mRNA isexpressed. If the polynucleotide in the sample is RNA, it is usuallyreverse transcribed to DNA. DNA samples or cDNA resulting from reversetranscription are usually amplified, e.g., by PCR. Depending on theselection of primers and amplifying enzyme(s), the amplification productcan be RNA or DNA. Paired primers are selected to flank the borders of atarget polynucleotide of interest. More than one target can besimultaneously amplified by multiplex PCR in which multiple pairedprimers are employed.

[0057] The target can be labeled at one or more nucleotides during orafter amplification. For some target polynucleotides (depending on sizeof sample), e.g., episomal DNA, sufficient DNA is present in the tissuesample to dispense with the amplification step. Preferably, thedetectable label is a luminescent label. Useful luminescent labelsinclude fluorescent labels (or fluorescent probe molecules),chemiluminescent labels, bio-luminescent labels, and colorimetriclabels, among others. Most preferably, the label is a fluorescent probemolecule such as a fluorescein, a rhodamine, a polymethine dyederivative, a phosphor, and so forth. Commercially available fluorescentlabels include, inter alia, fluorescein phosphoramidites such asFluoreprime (Pharmacia, Piscataway, N.J.), Fluoredite (Millipore,Bedford, Mass.) and FAM (ABI, Foster City, Calif).

[0058] Many alternatives to the detection of hybridized DNA byfluorescence exist. Most of the alternative techniques also involvemodification of capture or target or reporter DNA probes with reportergroups that produce a detectable signal. A few of these techniques basedon purely physical measurements do not require reporter groups. Thesealternative techniques are catalogued as follows: (1) Linear OpticalMethods including fluorescence, time modulated fluorescence,fluorescence quenching modulation, polarization selective fluorescence,absorption, specular reflectance, changes in index of refraction,ellipsometry, surface plasmon resonance detection, chemiluminescence,speckle interferometry and magneto-optic Kerr effect; (2) NonlinearOptical Methods including second harmonic generation, third harmonicgeneration, parametric mixing, optical heterodyne detection, phaseconjugation, soliton damping and optical Kerr effect; (3) Methods Basedon Thermal Effects including differential scanning calorimetry,multifrequency differential scanning calorimetry, and differentialthermal analysis; (4) Methods Based on Mass Changes including crystalmicrobalances, cantilever microbalances, surface acoustic waves andsurface Love waves; (5) Electrochemical Methods including amperometry,coulometry, voltammetry, electrochemiluminescence, charge transfer indonor-acceptor complexes and surface impedance spectroscopy; and (6)Radioactivity Detection Methods using labeled group.

[0059] More specifically, useful light scattering labels include largecolloids, and especially the metal colloids such as those from gold,selenium and titanium oxide. Radioactive labels include, for example,³²P. This label can be detected by a phosphoimager. Detection, ofcourse, depends on the resolution of the imager. Phosophoimagers areavailable having resolution of 50 microns. Accordingly, this label iscurrently useful with chips having features of at least that size.

[0060] When the target strand is prepared in single-stranded form as inpreparation of target RNA, the sense of the strand should of course becomplementary to that of the probes on the chip. This is achieved byappropriate selection of primers. The target is preferably fragmentedbefore application to the chip to reduce or eliminate the formation ofsecondary structures in the target. The average size of target segmentsfollowing hybridization is usually larger than the size of the probe onthe chip.

[0061] Substrate-Bound Oligonucleotide Arrays

[0062] Substrate-bound oligonucleotide arrays used in the assays of thisinvention typically include between about 5×10² and about 10⁸ featuresper square centimeter, or between about 10⁴ and about 10⁷ or betweenabout 10⁵ and 10⁶.

[0063] The construction of solid phase biopolymer arrays is welldescribed in the literature. See, e.g., Merrifield (1963) J. Am. Chem.Soc. 85: 2149-2154 (describing solid phase synthesis of, e.g.,peptides); Geysen et al. (1987) J. Immun. Meth. 102: 259-274 (describingsynthesis of solid phase components on pins). See, Frank and Doring(1988) Tetrahedron 44: 6031-6040 (describing synthesis of variouspeptide sequences on cellulose disks); Fodor et al. (1991) Science 251:767-777; Southern et al. (1992) Genomics 13: 1008-1017; Sheldon et al.(1993) Clinical Chemistry 39(4): 718-719 and Kozal et al. (1996) NatureMedicine 2(7): 753-759 (all describing arrays of biopolymers fixed tosolid substrates).

[0064] Preferably, the arrays are produced through spatially directedoligonucleotide synthesis. Methods for production of such arrays arewell known in the art and include any method of directing the synthesisof an oligonucleotide to a specific location on a substrate. Methods forspatially directed oligonucleotide synthesis include, withoutlimitation, light-directed oligonucleotide synthesis, microlithography,application by ink jet, microchannel deposition to specific locationsand sequestration with physical barriers.

[0065] In making a chip, the substrate and its surface preferably form arigid support on which the sample can be formed. The substrate and itssurface are also chosen to provide appropriate light-absorbingcharacteristics. For instance, the substrate may be functionalizedglass, Si, Ge, GaAs, GaP, SiO₂, SiN₄, modified silicon, or any one of awide variety of gels or polymers such as (poly)tetrafluoroethylene,(poly)vinylidenedifluoride, polystyrene, polycarbonate, or combinationsthereof. Other substrate materials will be readily apparent to thoseskilled in the art upon review of this disclosure. In a preferredembodiment the substrate is flat glass or silica. Surfaces on the solidsubstrate usually, though not always, are composed of the same materialas the substrate. Thus, the surface may be composed of any of a widevariety of materials, for example, polymers, plastics, resins,polysaccharides, silica or silica-based materials, carbon, metals,inorganic glasses, membranes, or any of the above-listed substratematerials. In one embodiment, the surface will be optically transparentand will have surface Si—OH functionalities, such as those found onsilica surfaces. Preferably, oligonucleotides are arrayed on a chip inaddressable rows and columns. Technologies already have been developedto read information from such arrays. The amount of information that canbe stored on each chip depends on the lithographic density which is usedto synthesize the wafer. For example, if each feature size is about 100microns on a side, each chip can have about 10,000 probe addresses(features) in a 1 cm² area.

[0066] Binding Assays

[0067] The methods described herein can also be used to facilitateELISAs or other binding assays wherein one binding partner isimmobilized on a solid support and the other is present in solution. Asdescribed above, methods for preparing arrays of biomolecules, includingpeptides, proteins, antibodies, and oligonucleotides are well known inthe art.

[0068] The assay mixture can also include a variety of other reagents,such as salts, buffers, neutral proteins, e.g., albumin, detergents, andthe like, which may be used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions, etc.Reagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, antimicrobial agents, and thelike, can also be used. The mixture components can be added in any orderthat provides for the requisite bindings.

[0069] Generally, incubation will be at temperatures used for suchbinding assay acids, for example, between about 20° C. and about 75° C.,e.g., about 25° C., about 30° C., about 35° C., about 40° C., about 45°C., about 50° C., about 55° C., about 60° C. or about 65° C. Preferably,a temperature of 37° C. -45° C. will be used.

[0070] Preferably, a temperature gradient of between about 5 and 25°C./mm, more preferably, between about 5 and 15° C./mm, and mostpreferably, a temperature gradient of about 10° C./mm will be used.

[0071] Incubation periods are likewise selected for optimal binding butalso minimized to facilitate rapid, high-throughput screening, and aretypically between 1 and 10 hours, preferably less than 5 hours, morepreferably less than 2 hours. For optimal high throughput applications,the reaction is carried out for between 0.1 and 4 hours, more typicallybetween about 0.5 and 1.5 hours.

[0072] After incubation, it may be desirable to separate any unboundtarget from the array. Typically, the separation step will include anextended rinse or wash or a plurality of rinses or washes. For example,the array may be washed several times with a washing solution, whichtypically includes those components of the incubation mixture that donot participate in specific binding such as salts, buffer, detergent,nonspecific protein, etc. In addition the temperature gradient may bereversed, so that thermophoretic forces assist in separating the unboundtarget from the array.

[0073] Detection can be effected in any convenient way. Frequently, oneof the components, generally, the target, comprises or is coupled to alabel. The assay component can be either directly labeled, i.e.,comprise or react to produce a detectable label, or indirectly labeled,i.e., bind to a molecule comprising or reacting to produce a detectablelabel. Labels can be directly attached to or incorporated into the assaycomponent or detection moiety by chemical or recombinant methods.

[0074] More specifically, the detectable labels used in the assays ofthe present invention, can be primary labels (where the label comprisesan element that is detected directly or that produces a directlydetectable element) or secondary labels (where the detected label bindsto a primary label, e.g., as is common in immunological labeling). Anintroduction to labels, labeling procedures and detection of labels isfound in Polak and Van Noorden (1997) Introduction toImmunocytochemistry, 2nd ed., Springer Verlag, N.Y. and in Haugland(1996) Handbook of Fluorescent Probes and Research Chemicals, a combinedhandbook and catalogue Published by Molecular Probes, Inc., Eugene,Oreg. Primary and secondary labels can include undetected elements aswell as detected elements. Useful primary and secondary labels in thepresent invention can include spectral labels such as fluorescent dyes(e.g., fluorescein and derivatives such as fluorescein isothiocyanate(FITC) and Oregon Green.TM., rhodamine and derivatives (e.g., Texas red,tetrarhodimine isothiocynate (TRITC), etc.), digoxigenin, biotin,phycoerythrin, AMCA, CyDyes.TM., and the like), radiolabels (e.g.,.sup.3 H, 125I, .sup.35 S, .sup. 14 C, .sup.32 P, .sup.33 P, etc.),enzymes (e.g., horseradish peroxidase, alkaline phosphatase etc.),spectral colorimetric labels such as colloidal gold or colored glass orplastic (e.g. polystyrene, polypropylene, latex, etc.) beads. The labelmay be coupled directly or indirectly to a component of the detectionassay (e.g., the detection reagent) according to methods well known inthe art. As indicated above, a wide variety of labels may be used, withthe choice of label depending on sensitivity required, ease ofconjugation with the compound, stability requirements, availableinstrumentation, and disposal provisions.

[0075] Preferred labels include those that use: 1) chemiluminescence(using horseradish peroxidase or luciferase) with substrates thatproduce photons as breakdown products as described above) with kitsbeing available, e.g., from Molecular Probes, Amersham,Boehringer-Mannheim, and Life Technologies/Gibco BRL; 2) colorproduction (using both horseradish peroxidase and/or alkalinephosphatase with substrates that produce a colored precipitate (kitsavailable from Life Technologies/Gibco BRL, and Boehringer-Mannheim));3) chemifluorescence using, e.g., alkaline phosphatase and the substrateAttoPhos (Amersham) or other substrates that produce fluorescentproducts, 4) fluorescence (e.g., using Cy-5 (Amersham), fluorescein, andother fluorescent tags); 5) radioactivity. Other methods for labelingand detection will be readily apparent to one skilled in the art.

[0076] Preferred enzymes that can be conjugated to detection reagents ofthe invention include, e.g., .beta.-galactosidase, luciferase, andhorseradish peroxidase. The chemiluminescent substrate for luciferase isluciferin. One embodiment of a chemiluminescent substrate for.beta.-galactosidase is 4-methylumbelliferyl-.beta.-D-galactoside.Embodiments of alkaline phosphatase substrates include p-nitrophenylphosphate (pNPP), which is detected with a spectrophotometer;5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT)and fast red/napthol AS-TR phosphate, which are detected visually; and4-methoxy-4-(3-phosphonophenyl) spiro[1,2-dioxetane-3,2′-adamantane],which is detected with a luminometer. Embodiments of horseradishperoxidase substrates include 2,2′azino-bis(3-ethylbenzthiazoline-6sulfonic acid) (ABTS), 5-aminosalicylic acid (5AS), o-dianisidine, ando-phenylenediamine (OPD), which are detected with a spectrophotometer;and 3,3,5,5′-tetramethylbenzidine (TMB), 3,3′diaminobenzidine (DAB),3-amino-9-ethylcarbazole (AEC), and 4-chloro-1-naphthol (4C1N), whichare detected visually. Other suitable substrates are known to thoseskilled in the art. The enzyme-substrate reaction and product detectionare performed according to standard procedures known to those skilled inthe art and kits for performing enzyme immunoassays are available asdescribed above.

[0077] In general, a detector which monitors a particular label is usedto detect the label. Typical detectors include spectrophotometers,phototubes and photodiodes, microscopes, scintillation counters,cameras, film and the like, as well as combinations thereof. Examples ofsuitable detectors are widely available from a variety of commercialsources known to persons of skill. Commonly, an optical image of asubstrate comprising bound labeling moieties is digitized for subsequentcomputer analysis.

[0078] Most typically, binding of the target molecule to thecorresponding immobilized binding partner is measured by quantitatingthe amount of label fixed to the solid support by binding of thedetection reagent. Typically, presence of a modulator during incubationwill increase or decrease the amount of label fixed to the solid supportrelative to a control incubation which does not comprise the modulator,or as compared to a baseline established for a particular assay type.Means of detecting and quantitating labels are well known to those ofskill in the art. Thus, for example, where the label is a radioactivelabel, means for detection include a scintillation counter orphotographic film as in autoradiography. Where the label is opticallydetectable, typical detectors include microscopes, cameras, phototubesand photodiodes and many other detection systems which are widelyavailable.

[0079] The references discussed herein are provided solely for theirdisclosure prior to the filing date of the present application and areeach incorporated herein by reference. Nothing herein is to be construedas an admission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention.

[0080] Various modifications and variations of the described method andsystem of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes are carrying out theinvention which are obvious to those skilled in the art are intended tobe within the scope of the following claims.

what is claimed is:
 1. A method for performing a hybridization assaybetween a target nucleic acid molecule and an oligonucleotide array, thearray comprising a surface to which are covalently attachedoligonucleotide probes with different, known sequences, at discrete,known locations, the method comprising the steps of: incubating thearray with a hybridization mixture comprising the target underthermophoretic conditions; and determining the identity of probes towhich the target has hybridized.
 2. The method of claim 1 wherein thetarget further comprises a detectable label.
 3. The method of claim 2wherein the label is a fluorescent probe molecule.
 4. The method ofclaim 3 wherein the fluorescent probe molecule is fluorescein.
 5. Themethod of claim 1 wherein the array has a density of at least tenthousand features per square cm.
 6. The method of claim 5 wherein thearray has a density of at least one hundred thousand features per squarecm.
 7. The method of claim 6 wherein the array has a density of at leastone million features per square cm.
 8. The method of claim 1 whereinthermophoretic conditions comprise the application of a temperaturegradient perpendicular to the array surface whereby the target is drivento the array surface.
 9. The method of claim 8 wherein the array surfaceis vertical and the temperature gradient is horizontal.
 10. The methodof claim 8 wherein the array surface is horizontal and the temperaturegradient is vertical.
 11. The method of claim 8, further comprising thestep of: reversing the temperature gradient, whereby any unhybridizedtarget is driven away from the array surface.
 12. The method of claim 8,wherein the temperature gradient is between about 5 and 25° C./mm. 13.The method of claim 8, wherein the hybridization mixture furthercomprises an isostabilizing agent.
 14. A method for performing ahybridization assay between a target nucleic acid molecule and anoligonucleotide array, the array comprising a surface to which arecovalently attached oligonucleotide probes with different, knownsequences, at discrete, known locations, wherein such probes have beencontacted with a hybridization mixture comprising the target nucleicacid molecule, the method comprising the steps of: applying atemperature gradient to the array surface whereby any unhybridizedtarget is driven away from the array surface; and determining theidentity of probes to which the target has hybridized.
 15. A method forperforming a binding assay between a target molecule and an array, thearray comprising a surface to which are covalently attached a pluralityof binding partners with different, known sequences, at discrete, knownlocations, the method comprising the steps of: incubating the array witha mixture comprising the target under thermophoretic conditions; anddetermining the identity of binding partners to which the target hasbound.
 16. The method of claim 15, wherein the target further comprisesa detectable label.
 17. An apparatus for performing a hybridzationassay, comprising a container connected to at least one temperaturecontrol blocks in a heat-conducting fashion, such that a temperaturegradient is produced.
 18. The apparatus of claim 17, wherein thecontainer is connected to two temperature control blocks in aheat-conducting fashion.
 19. The apparatus of claim 17, furthercomprising an inlet port and an outlet port.
 20. The apparatus of claim17, further comprising an aperture to permit optical access to thecontainer.